title: Encoded multisite two-photon microscopy.
abstract: The advent of scanning two-photon microscopy (2PM) has created a fertile new avenue for noninvasive investigation of brain activity in depth. One principal weakness of this method, however, lies with the limit of scanning speed, which makes optical interrogation of action potential-like activity in a neuronal network problematic. Encoded multisite two-photon microscopy (eMS2PM), a scanless method that allows simultaneous imaging of multiple targets in depth with high temporal resolution, addresses this drawback. eMS2PM uses a liquid crystal spatial light modulator to split a high-power femto-laser beam into multiple subbeams. To distinguish them, a digital micromirror device encodes each subbeam with a specific binary amplitude modulation sequence. Fluorescence signals from all independently targeted sites are then collected simultaneously onto a single photodetector and site-specifically decoded. We demonstrate that eMS2PM can be used to image spike-like voltage transients in cultured cells and fluorescence transients (calcium signals in neurons and red blood cells in capillaries from the cortex) in depth in vivo. These results establish eMS2PM as a unique method for simultaneous acquisition of neuronal network activity.
authors.affiliation: Institut National de la Santé et de la Recherche Médicale U603, Paris 75006, France.
authors.forename: Mathieu
authors.initials: M
authors.lastname: Ducros
authors.forename: Yannick
authors.initials: Y
authors.lastname: Goulam Houssen
authors.forename: Jonathan
authors.initials: J
authors.lastname: Bradley
authors.forename: Vincent
authors.initials: V
authors.lastname: de Sars
authors.forename: Serge
authors.initials: S
authors.lastname: Charpak
mesh_terms: D000465:Algorithms; D000818:Animals; D001783:Blood Flow Velocity; D001921:Brain / Q000098:blood supply / Q000166:cytology / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling; D057809:HEK293 Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000295:instrumentation / Q000379:methods; D050866:Liquid Crystals; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal / Q000295:instrumentation / Q000379:methods; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation* / Q000379:methods*; D015233:Models, Statistical; D009474:Neurons / Q000378:metabolism*; D015203:Reproducibility of Results; D013997:Time Factors
chemical_list: D002118:Calcium
keywords: multiplexing; multipoint; scanless two-photon microscopy; voltage-sensitive dyes